Chapter 9 Commercial Potential of Microbial Inoculants for ... · chemicals in ShB management is...

Chapter 9

Commercial Potential of Microbial Inoculants

for Sheath Blight Management and Yield

Enhancement of Rice

K. Vijay Krishna Kumar, M.S. Reddy, J.W. Kloepper, K.S. Lawrence,

X.G. Zhou, D.E. Groth, S. Zhang, R. Sudhakara Rao, Qi Wang,

M.R.B. Raju, S. Krishnam Raju, W.G. Dilantha Fernando, H. Sudini, B. Du,

and M.E. Miller

K.V.K. Kumar

Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA

and

Acharya N G Ranga Agricultural University, Hyderabad, India

M.S. Reddy (*), J.W. Kloepper, and K.S. Lawrence

Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA

e-mail: [email protected]

X.G. Zhou

Agri-Life Research and Extension Center, Texas A and M University, College Station, TX, USA

D.E. Groth

LSU AgCenter, Rice Research Station, Rayne, LA, USA

S. Zhang

Tropical REC, University of Florida, Homestead, FL, USA

R.S. Rao

Acharya N G Ranga Agricultural University, Hyderabad, Andhra Pradesh, India

Q. Wang

China Agricultural University, Beijing, China

M.R.B. Raju and S.K. Raju

Andhra Pradesh Rice Research Institute, Maruteru, Andhra Pradesh, India

W.G.D. Fernando

Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada

H. Sudini

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Andhra

Pradesh, India

B. Du

Department of Microbiology, Shandong Agricultural University, Taian, Shandong Province,

China

M.E. Miller

Department of Biological Sciences, Auburn University, Auburn, AL, USA

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems,DOI 10.1007/978-3-642-18357-7_9, # Springer-Verlag Berlin Heidelberg 2011

237

9.1 Introduction

Rice (Oryza sativa L.) is an important staple food crop for a larger part of the

world’s population and is produced around the globe. Global rice production was

approximately 680 million tons in 2009. More than 90% of rice is produced in Asia,

with China and India being the lead producers. The other major rice-producing

countries are Indonesia, Bangladesh, Vietnam, Thailand, Myanmar, Philippines,

Brazil, and Japan (Table 9.1). Rice production in the USA, which started 300 years

ago, now has an annual production of 9.2 million tons. Major rice-producing states

of the USA are Arkansas, California, Louisiana, Mississippi, Missouri, and Texas.

The forecasted increase in global population in the coming years is demanding a

need for increase in productivity of rice, although there is only a limited scope for

expansion of crop-growing area especially in densely populated countries such as

Asia (Meunchang et al. 2006). Use of chemical fertilizers for enhancing rice

production is a common practice. However, indiscriminate use of chemical fertili-

zers to increase grain yields in rice has several concerns such as leaching of

fertilizers into ground water, change of microbial balance in soil–root-ecosystem,

increased susceptibility of the crop to pests and diseases, and acidification or

alkalization of soils.

Rice production is affected by many biotic and abiotic stresses including fungal

pathogens that attack the crop from seeding to harvest and cause severe yield

losses. Seed-borne pathogens often reduce the germination and inflict qualitative

and quantitative yield losses (Haque et al. 2007). Among important fungal diseases,

blast (Magnaporthe oryzae, formerly M. grisea or Pyricularia oryzae), sheathblight (Rhizoctonia solani AG 1-1A), brown spot (Bipolaris oryzae), sheath rot

(Acrocylindrium oryzae), stem rot (Sclerotium oryzae), and bakane (Gibberellafujikuroi) cause severe yield losses in rice. Major bacterial diseases include bacte-

rial leaf blight (Xanthomonas campestris pv. oryzae) and bacterial leaf streak

(X. campestris pv. oryzicola) (Bangura and John 1991). Important viral diseases

include tungro, grassy stunt, ragged stunt, yellow dwarf, orange leaf, and hoja

Table 9.1 Production details of major rice-producing countries in the worlda

Rank Country Rice production (million tons)

1 China 187.40

2 India 144.57

3 Indonesia 57.15

4 Bangladesh 43.06

5 Viet Nam 35.94

6 Thailand 32.10

7 Myanmar 31.45

8 Philippines 16.24

9 Brazil 11.06

10 Japan 10.89aFAOSTAT 2007

238 K.V.K. Kumar et al.

blanca. Other important ones include diseases caused by nematodes such as white

tip (Aphelenchoides besseyi) and ufra (Ditylenchus angustus) (Datta 1981).Sheath blight (ShB) is an economically significant disease of rice in all growing

areas of the world. Yield losses of up to 50% are reported when susceptible varieties

are grown (Prasad and Eizenga 2008). Soil bacteria in rice ecosystems typically

exert a significant fungistatic effect on mycelia and sclerotia of the ShB pathogen

(Luo et al. 2005). Effective management of ShB with PGPR application has been

reported (Mew and Rosales 1986; Vasantha Devi et al. 1989; Kanjanamaneesathian

et al. 1998); however, the field results were not consistent due to varying reasons.

This review focuses on recent developments in the management of rice ShB with

PGPR. The topics covered in the chapter include PGPR application in rice, green-

house, and field efficacy of PGPR and the scope of applying them in conjunction

with chemical fungicides under integrated disease management system (IDM) of

ShB. The overall goal of this chapter is to introduce the multistep process that leads

to the development of a new microbial inoculant product and its use and to outline

the beneficial strategies specifically for ShB disease management of rice. In addi-

tion, it attempts to define the major efforts under way to help stimulate the process.

Because product development is integrally related to several tasks including intel-

lectual property issues and to regulatory and liability concerns, these topics are also

included. Data on product development for rice ShB management are not system-

atically available. We have, therefore, used information based on our own research

efforts and, when possible, made comparisons.

9.2 Symptomatology

Initial ShB symptoms appear on lower rice leaf sheaths when the crop is in late

tillering or early internode elongation phase. These lesions appear as green–grey

water soaked at 0.5–3 cm below the collar region as circular, oblong, or ellipsoid

and about 1 cm long. As the disease progresses, the lesions expand with bleached

appearance and a brown border. Under favorable conditions (95% relative humidity

and temperature of 28–32�C), the disease spreads by runner hyphae to upper parts

of plants including leaf blades (Fig. 9.1a). The pathogen also infects the panicle

(Fig. 9.1b) and causes chaffiness of lower grains (Lee and Rush 1983).

9.3 Disease Cycle

The pathogen survives from one crop season to another as sclerotia and mycelia

in plant debris and also through weed hosts in tropical environments (Kobayashi

et al. 1997). In temperate regions, the primary source of inoculum is sclerotia

produced in previous rice crops (Kozaka 1961). The sclerotia float in water during

field preparation and attack newly planted crop. The pathogen produces lesions on

9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 239

leaf sheaths and leaf blades. The disease is more aggressive when the crop advan-

ces to the reproductive phase, and the pathogen also infects the rice panicles. New

sclerotia are produced as the lesions mature and these sclerotia drop into the soil

during harvesting, perpetuate, and infect a newly planted crop in the next season

(Suparyono et al. 2003).

9.4 Use of Microbial Inoculants

Currently, ShB is managed through cultural and chemical control methods. Mostly,

disease management is through use of systemic and non-systemic fungicides. Most

widely used fungicides include azoxystrobin, hexaconazole, propiconazole, tebu-

conazole, carbendazim, trifloxystrobin, validamycin, and jinggangmycin. Use of

chemicals in ShB management is creating concerns over environmental pollution,

escalated costs, and pathogen resistance to chemicals. Biological control is a viable

alternative in ShB disease management. However, the use of biocontrol agents in

managing rice diseases is still at its infancy due to varying reasons. A successful

bioagent, when applied to rice ecosystem, should be able to survive, establish,

proliferate, and control target pathogens. Fungal and bacterial biocontrol agents

have been used for control of rice diseases. The popularly used fungal bioagents

against ShB include Trichoderma spp. and Gliocladium spp. These bioagents were

applied either as seed treatment, root dip, or foliar spray (Nagaraju et al. 2002). The

other effective fungal bioagent is Helminthosporium gramineum that produces

a toxin called “ophiobolin.” The toxin is effective in reducing ShB incidence

under field conditions (Duan et al. 2007). The prevailing anaerobic conditions

in rice are unfavorable for the fungal bioagents to survive, establish, and proliferate

in the soil.

Rice ecosystems are rich in bacteria (Yin and Mew unpublished data; Mew et al.

2004). They also have greater adaptability to rice ecosystems compared to fungal

antagonists. Of them, plant growth-promoting rhizobacteria (PGPR) have been

Fig. 9.1 Sheath blight symptoms on rice leaf blades and panicle (a) leaf blades. (b) Formation of

sclerotia on panicle


used in controlling rice diseases. Besides, these PGPR also contribute to enhanced

growth of the seedlings, induction of systemic resistance against diseases and

thereby increases yields (Pathak et al. 2004). Bacterial strains of the genera such

as Aeromonas, Azoarcus, Azospirillum, Azotobacter, Arthobacter, Bacillus, Clos-tridium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, and Serratiawere identified as PGPR (Tripathi et al. 2005; Raj et al. 2004; Dey et al. 2004;

Jaizme-vega et al. 2004; Joo et al. 2004; Bonaterra et al. 2003; Cezon et al. 2003;

Esitken et al. 2003; Garica et al. 2003; Munir et al. 2003; Kokalis-Burelle et al.

2002; Khalid et al. 2003; Murphy et al. 2003; Preeti et al. 2002; Gupta et al. 1995;

Bertand et al. 2001; Hamaoui et al. 2001; NandaKumar et al. 2001b; Pan et al. 1999;

Arndt et al. 1998; De Freitas et al. 1997; Shishido et al. 1996; Babalola et al. 2003;

Mirza et al. 2001; Podile and Kishore 2006). In addition to enhancement in plant

growth, PGPR were also contributed to increase N uptake, phytohormone synthesis,

phosphate solubilization, and acquisition of ferric iron through production of side-

rophores (Lalande et al. 1989; Glick 1995; Bowen and Rovira 1999).

Use of PGPR in rice to control major diseases and to enhance yields was earlier

reported (Lucas et al. 2009). A variety of beneficial bacteria were found to colonize

the rhizosphere and aerial parts of rice. Nitrogen-fixing activity and indoleacetic

acid (IAA) production was detected in roots and submerged shoots of field-grown

rice due to these beneficial bacteria (Mehnaz et al. 2001). Rhizosphere bacterial

isolates of rice have an excellent potential of producing biofertilizers. Inoculation

of PGPR in rice increased total dry weight of plants, total N and P uptake through

N fixation, P solubilization capacity, and IAA production (Meunchang et al. 2006).

Use of biofertilizers in cereals was found to significantly increase plant growth and

yields (Boddey et al. 1986; Fages 1994; Kapulnik et al. 1981; Kennedy and Tchan

1992; Pereira et al. 1988). Frequent rhizosphere colonizers of cereal crops and

grasses include N-fixing bacteria such as Azospirillum, Acetobacter, Azoarcus,Herbaspirillum spp. (Baldani et al. 1986; Bally et al. 1983; Bilal et al. 1990;

Dobereiner and Day 1976; Gillis et al. 1989; Reinhold-Hurek et al. 1993), Aero-monas, and Enterobacter spp. (Mehnaz et al. 2001).

9.4.1 Mode of Delivery

Field efficacy of a PGPR strain partly depends on the method of delivery. PGPR

and their formulations are generally delivered as seed treatment, soil amendment,

or root dip in bacterial suspensions prior to transplanting. Other important

methods also include foliar spray or through drip irrigation in different crops

(Podile and Kishore 2006). Success of the PGPR strain is dependent on under-

standing the use of specific delivery system and its advantages over other

methods. In rice, PGPR is delivered through seed, as soil amendment, seedling

dip, and foliar spray, and through combinations of these methods. Against rice

ShB, the popular delivery systems are through seed, soil, and foliar applications

(Nakkeeran et al. 2005).


9.4.1.1 Seed Treatment

An ideal bacterial antagonist when treated to seed should colonize the rhizosphere

during seed germination (Weller 1983), and several application methods can be

used to accomplish this. Treating seeds with different PGPR was found to be highly

effective in managing rice ShB disease. Seed coating of P. fluorescens (B41) wasfound to be comparatively more effective than soil drenching and foliar sprays

against ShB under greenhouse conditions (Kazempour 2004). Seed bacterization of

Pseudomonas strain GRP3 followed by root dipping resulted in ShB reduction in

rice up to 46% (Pathak et al. 2004). Seed treatment with PGPR mixtures also

resulted in effective ShB management. Soaking rice seeds in P. fluorescensmixture

of strains PF1 and PF2 at 108 cfu g�1 for 24 h were effective in reducing ShB

incidence under field conditions (Nandakumar et al. 2001a). Seed bacterization

with fluorescent Pseudomonads such as P. fluorescens and P. putida V14i was

highly effective in reducing ShB severities by 68 and 52%, respectively, in seed bed

and field experiments (Malarvizhi 1987) due to protection of the plants from

infection. Subsequent planting in the same field after planting the first crop, in

which the seeds were treated with bacteria, also showed reduced ShB severity

(Mew and Rosales 1986). Seed treatment with peat-based formulation of P. fluor-escens (PfALR2) at the rate of 20 g kg�1 resulted in ShB disease control effectively

under greenhouse and field conditions (Rabindran and Vidhyasekaran 1996).

Induced systemic resistance, plant growth promotion, and sheath blight control

was observed by treating rice seeds with three isolates of Pseudomonas aeruginosa.The biocontrol agents were also found effective in reducing blast and brown spot

diseases in rice due to increased accumulation of salicylic acid and pathogenesis-

related peroxidases (Saikia et al. 2006).

9.4.1.2 Seedling Dip

The ShB pathogen is soil-borne, attacks the rice seedlings, and establishes host–

pathogen relationship by root entry (Nakkeeran et al. 2005). Seedling root dip

treatment of rice prior to transplanting for a period of 2 h in talc-based formulations

of PGPR mixtures at 20 g/L reduced ShB incidence effectively (Nandakumar et al.

2001a). Earlier, seedling root dip in a talc-based formulation of P. fluorescensbefore transplantation into main field suppressed ShB disease and improved

grain yields (Rabindran and Vidhyasekaran 1996). A novel application method of

B. megateriummultiplied in empty fruit bunches (EFB) as carrier was reported. The

rice seedlings when treated with bacterial inoculum multiplied in EFB carrier had

significantly enhanced plant height, number of roots, and dry matter of root and

shoot. The method offered a scope of developing new delivery system and granula-

tion of bioinoculants for effective control of diseases as well as for enhancing grain

yields (Al-Taweil et al. 2009).


9.4.1.3 Soil Application

Soil application of PGPR has also been reported to be an effective method of

controlling soil-borne diseases of rice (Rabindran and Vidhyasekaran 1996). For

effective suppression of ShB, the population thresholds of the antagonist in soil

should be higher than 1 � 106 cfu g�1 during early stages of infection by R. solani(Li et al. 2003). Effective management of ShB is feasible only when the applied

bioagents survive, establish, proliferate, and control pathogen populations in soils.

A strain of B. licheniformis (CHM1) isolated from rice fields was found to be highly

effective in protecting rice seedlings from ShB disease as well as in plant-growth

promotion when applied as soil drenching around root zone (Wang et al. 2009). Soil

application of peat formulation of P. fluorescens (PfALR2) effectively controlled

ShB disease under greenhouse and field conditions (Rabindran and Vidhyasekaran

1996). Broadcasting of talc-based formulation mixtures of Pf1 and Pf7 at 30 days of

transplanting of rice seedlings reduced ShB and increased grain yields significantly

under field conditions (Nandakumar et al. 2001a). The population levels of PGPR in

rice fields are an important factor for effective control of ShB disease. Mixing the

potting soil with bacterial suspensions of different P. aeruginosa mutants coupled

with a soil drench at a concentration of 5 � 107 cfu g�1 elicited ISR in rice

seedlings to blast and ShB diseases under greenhouse conditions (Vleesschauwer

and Hofte 2005).

9.4.1.4 Foliar Application

Survival rates and application efficiencies of PGPR as foliar sprays against plant

diseases is generally affected by variations in microclimate. Nutrient concentrations

of amino acids, organic acids, and sugars that exude through lenticels, stomata, and

hydathodes vary in the phyllosphere (Nakkeeran et al. 2005). The efficacy of PGPR

against ShB under greenhouse and field conditions is dependent on time of appli-

cation. Spraying of P. fluorescens at 7 days before pathogen inoculation resulted ineffective ShB reduction (59–64%) over simultaneous application at 7 days after

inoculation. Further, grain yields and 1,000 grain weight were also enhanced with

the prophylactic sprays (Rajbir Singh and Sinha 2005). Commercial formulations of

P. fluorescens (Ecomonas and Florezen P) when sprayed three times at 10-day

interval after disease initiation under field conditions resulted in ShB control by

14–38% besides significant increase in grain yields (Vijay Krishna Kumar et al.

2009). Foliar sprays with floating pellet formulation of B. megaterium were effec-

tive in rice ShB suppression under greenhouse conditions (Wiwattanapatapee et al.

2007). Spraying of antifungal metabolites of Streptomyces spp. (SPM5C-2) at the

rate of 500 mg ml�1 significantly decreased ShB and blast disease development by

82 and 76%, respectively, under greenhouse conditions (Prabavathy et al. 2006).

Broadcasting of floating pellets formulation and spraying of water-soluble formu-

lation of B. megaterium resulted in effective control of rice ShB disease under

greenhouse and field conditions (Kanjanamaneesathian et al. 2007).


9.4.1.5 Multiple Delivery Systems

Protection of spermosphere, rhizosphere, and phylloplane from infection courts of

plant pathogens through multiple delivery systems of PGPR offers a comprehensive

means of plant disease management (Nakkeeran et al. 2005). Talc-based formula-

tions of two P. fluorescens strains (PF1 and PF7) when applied through seed, root,

soil, and foliar sprays significantly reduced ShB and pest (leaf-folder) incidence in

rice under greenhouse and field conditions. The bacterial mixture performed better

than individual strains with a reduction of 62% of ShB and 47–56% of leaffolder

incidence (Radja Commare et al. 2002). Combined applications of P. fluorescensstrains (PF1, FP7, and PB2) as bacterial suspensions or as talc-based formulations

through seed, root, foliar, and soil application significantly reduced the ShB inci-

dence (45%) under greenhouse and field conditions over their individual applica-

tions. Further, a significant increase in yield was obtained with application of

mixtures over their individual applications. Fluorescent Pseudomonas application(PF1 and FP7) either as a suspension or talc-based formulation through seed, root,

soil, and foliar means effectively reduced rice ShB incidence and promoted plant

growth and grain yields (Nandakumar et al. 2001a). Similar results on ShB disease

suppression and enhanced yields were reported with peat-based formulations of

P. fluorescens (PfALR2) as seed treatment, root treatment, soil application, and

foliar spraying. Further, the efficacy of combined application methods was compa-

rable with fungicide treatments (Rabindran and Vidhyasekaran 1996).

9.4.2 Formulations

A formulated PGPR should ideally possess high rhizosphere competence, plant

growth promotion, ease for large-scale multiplication, wide range of plant disease

control, consistent in disease control, and compatible with environment and other

rhizobacteria (Nakkeeran et al. 2005). Besides, the bacterial inoculants should be

able to tolerate desiccation, heat, oxidizing agents, and UV radiations (Jeyarajan

and Nakkeeran 2000). The formulated product should meet the important criteria

such as satisfactory shelf life, non-phytotoxic nature, water solubility, ability to

withstand environmental fluctuations and compatibility with other agrochemicals.

Besides, it should be cost-effective with ready availability of carriers at a cheaper

rate and should not impart mammalian toxicity (Nakkeeran et al. 2005; Jeyarajan

and Nakkeeran 2000).

The carrier materials used in PGPR formulations are broadly categorized into

organic and inorganic ones. The commonly used organic carriers are peat, turf, talc,

lignite, kaolinite, pyrophyllite, zeolite, montmorillinite, alginate, pressmud, sawdust,

and vermiculite (Nakkeeran et al. 2005). In general, PGPR survive longer in carriers

with smaller particle sizes than in those with larger particle sizes. Carriers with

smaller size will have more surface area that enables increased resistance to desicca-

tion of PGPR through increased coverage of bacterial cells (Dandurand et al. 1994).


Commonly available PGPR formulations are talc formulations, peat formula-

tions, press mud formulations, vermiculite formulations (Nakkeeran et al. 2005),

water-soluble granular formulations, liquid formulations, floating pellet formula-

tions, and formulations with EFB as carriers. Details of different PGPR formula-

tions that exhibited effective control of rice ShB disease under greenhouse or field

conditions are given in Table 9.2.

9.4.3 Shelf life

Effective disease control by PGPR is possible only when the formulated product

delivers a sufficient number of viable cells. So, determining the shelf life and

viability of a commercial bio-product is a crucial step. The shelf life of PGPR in

the formulated product is dependent on the type of carrier material used. Talc is an

excellent carrier material for PGPR with low moisture equilibrium, relative hydro-

phobicity, reduced moisture absorption, and chemical inertness (Nakkeeran et al.

2005). The population levels of PGPR (fluorescent Pseudomonads) did not decline

in talc powder with 20% xanthan gum after storage for 2 months at 4�C (Kloepper

and Scroth 1981).

Vermicompost is comparatively a better carrier material than lignite for bioino-

culants with high nitrogen, phosphorus, potassium, copper, manganese, and iron

besides possessing an ideal pH. The shelf life of vermicompost-based formulations

is greater than that of lignite-based ones. The population levels of B. megateriumand P. fluorescens were very high (7.6 � 108 and 1 � 108 cfu g�1 of dry weight,

respectively) at the end of 360 days when vermicompost was used as carrier

(Gandhi and Saravanakumar 2009).

The shelf life of peat-based formulations depends on the availability of good

quality peat. Heat sterilization of peat results in release of toxic substances that are

detrimental to bacteria thus affecting their population levels in the formulation

(Bashan 1998). The population levels of P. fluorescens (2.8 � 106 cfu g�1) in peat-

based formulation was maintained up to 8 months (Vidhyasekaran and Muthamilan

1995), whereas the shelf life of P. chlororaphis and B. subtilis were more than 6

months (Kavitha et al. 2003; and Nakkeeran et al. 2004). The use of press mud and

vermiculite-based PGPR formulations are also in practice. The viability of Azos-pirillum spp. in press mud formulation is higher than in lignite (Muthukumarasamy

et al. 1997) whereas in vermiculite, its viability is retained up to 10 months (Saleh

et al. 2001).

9.4.4 Root Colonization

Of different soil microbial populations, bacteria residing in the rhizosphere are the

most beneficial. Bacterial communities in the rhizosphere vary in different root


Table

9.2

CurrentlyusedPGPRform

ulationsagainstrice

sheath

blightdisease

PGPRstrain

Typeofform

ulation

Methodofapplication

References

Bacillusmegaterium

EFB(empty

fruitbunches)powder

Seedlingdip

Al-Taw

eilet

al.(2009)

BacilluslicheniformisCHM1

Bacterial

cellsuspension

Soilapplication

Wanget

al.(2009)

Bacillusmegaterium

(No16)

Floatingpellets

Broadcasting

Kanjanam

aneesathianet

al.(2007)

Bacillusmegaterium

(No16)

Water

soluble

granules

Foliar

spray

Kanjanam

aneesathianet

al.(2007)

Pseudo

mona

sflu

orescens(Pf1

andFP7)

Bacterial

suspension/talc-based

Seed+root+soil+foliar

Nandakumar

etal.(2001a)

Pseud

omon

asfluo

rescensand

Pseudo

mona

s.Aerug

inosa

Bacterial

cellsuspension

Seed+root+soil

Vleesschauwer

andHofte(2005)

Pseud

omon

asfluo

rescens(PfA

LR2)

Peatbased


RabindranandVidhyasekaran

(1996)

Pseudo

mona

sflu

orescens(Pf1

andFP7)

Talc-based


Radja

Commareet

al.(2002)

Beneficial

bacteria(N

F1,NF3,NF52,

NF49,CT6-37,andW23)

Bacterial

cellsuspension

Foliar

spray

Lai

Van

Eet

al.(2001)

Fluorescentandnon-fluorescent

Pseudomonads

Bacterial

cellsuspension

Seedtreatm

ent

Mew

andRosales(1986);Vasantha

Deviet

al.(1989)

Bacillusmegaterium

Floatingpellets

Broadcast

+spray

Wiwattanapatapee

etal.(2007)

Pseudo

mona

sflu

orescens

Talc-based

Foliar

spray

Vijay

KrishnaKumar

etal.(2009)

Pseudo

mona

saeruginosa

Bacterial

cellsuspension

Foliar

spray

Saikia

etal.(2006)

Antagonisticbacteria

Bacterial

cellsuspension

Seed+foliar

Chen

etal.(1996)


zones and their composition can be altered by changes in root exudate composition

(Yang and Crowley 2000). Root exudates of rice plants were found to exert

a positive influence on the motility of these bacteria toward plant roots (Bacilio-

Jiminez 2003). Earlier studies indicated that the rhizosphere isolates of rice were

able to induce IAA production and have phosphate solubilization capacity. Further,

these PGPR isolates were found to promote seed germination, root length, plant

height, and dry matter production of shoot and roots in rice (Ashrafuzzaman et al.

2009). Application of bio-inoculants was found to enhance rice growth through

production of total sugars, reducing sugars, amino nitrogen content, PGP sub-

stances in the root exudates, and biological nitrogen fixation. The microbial con-

sortium viz., Azospirillum lipoferum-Az204, B. megaterium var. phosphaticum, andP. fluorescens Pf1 when applied to rice improved the colonization potential,

sustainability within the inoculants, and enhanced plant growth when compared

to their application individually (Raja et al. 2006).

Mirza et al. (2006) reported a nitrogen-fixing, phytohormone-producing Pseu-domonas isolate (strain K1) that had a capacity of fixing nitrogen in inoculated rice

plants and its efficacy was comparable to non-Pseudomonas nitrogen-fixing PGPR.Use of PGPR also alleviates zinc deficiency in rice plants. Zinc deficiency is a

serious problem in rice production (Anon 1993). Inoculation of rice fields with

PGPR had a significant positive impact on root length (54% increase), root weight

(74%), root volume (62%), root area (75%), shoot weight (23%), panicle emer-

gence index (96%), and Zn mobilization efficiency, thereby reducing the cost

incurred in the application of chemical Zn fertilizers (Muhammad et al. 2007).

Application of diazotrophs such as Rhizobium leguminosarum bv. trifolii (E11),Rhizobium spp. (IRBG74), and Bradyrhizobium sp. IRBG271 in lowland rice fields

enhanced N, P, and K uptake by 10–28% due to rhizobial inoculation. In addition,

the uptake of Fe was enhanced by 15–64%. Further, the growth promotion in rice

was due to changes in growth physiology or root morphology rather than biological

nitrogen fixation (BNF) (Biswas et al. 2000).

9.5 Sheath Blight Management

In rice, PGPR offer a promising means of controlling plant diseases besides

contributing to the plant resistance, growth, and grain yields (Mew and Rosales

1992). Of different PGPR, fluorescent Pseudomonads and Bacillus spp. group of

bacteria are widely used against ShB. Their application promotes plant growth by

direct and indirect mechanisms. Direct growth promotion is due to production of

phytohormones, solubilization of phosphates, increased uptake of iron through

production of siderophores, and volatile metabolites. Indirect way of plant growth

promotion is due to mechanisms of antibiosis, competition for space and nutrients,

parasitism or lysis of pathogen hyphae, inhibition of pathogen-produced enzymes

or toxins, and through induced systemic resistance (ISR). The ISR in rice against

ShB is either due to enhanced chitinase or peroxidase activity (Nandakumar et al.


2001b). However, no correlation was observed between chitinase production and

ShB suppression (Thara and Gnanamanickam 1994). Strains of P. fluorescens werefound to produce siderophores, volatile metabolites, extracellular secretions, and

antibiotics that were inhibitory to ShB pathogen. Further, the strains reduced

germination and caused lysis of sclerotia (Kazempour 2004). Rhizosphere isolates

of P. fluorescens produced b-1,3-glucanase, salicylic acid, and HCN, and a signifi-

cant relationship was observed between antagonism of the bacterium and the

production of these substances (Nagarajkumar et al. 2004).

Bacillus spp. are endospore-producing gram-positive bacteria, and some strains

have been used in biocontrol of rice diseases. Strains of B. subtilis and B. mega-terium exhibit inhibition of Rhizoctonia solani (Luo et al. 2005). The fermented

product of Bacillus (Drt-11) is highly inhibitory to the sclerotial germination,

hyphal growth, and colony diameter besides enhancing rice seedling growth

(Chen and Hui 2006). Strains of Bacillus produce a thermo and proteinase – stable

antagonistic substance (P1) that is effective against rice ShB and blast pathogens

(He et al. 2002). The B. subtilis (AUBS1) strain produces phenylalanine ammonia-

lyase (PAL), peroxidase (PO), and certain pathogenesis-related (PR) proteins in

rice leaves when applied against ShB disease. Accumulation of thaumatin-like

proteins, glucanases, and chitinases are the other important substances in plants

against ShB by these bioagents (Jayaraj et al. 2004). The other promising bacteria

against rice ShB include Streptomyces spp. and Serratia marcescens. The antifun-gal metabolites of Streptomyces spp. (PM5, SPM5C-1, and SPM5C-2) were highly

effective against mycelial growth of rice ShB and blast pathogens under in vitro

conditions. Greenhouse studies revealed that spraying of the strain SPM5C-2 at

500 mg ml�1 significantly reduced ShB and blast diseases by 82 and 76%, respec-

tively (Prabavathy et al. 2006). Culture filtrates of S. marcescens exhibited

enhanced reduction of sclerotial viability of ShB pathogen, when applied with

reduced doses of fungicides such as flutolanil, pencycuron, and validamycin

(Someya et al. 2005).

In order to identify a potential biocontrol agent, researchers have been spending

their time on several microbes in areas of isolation, identification, and purification

which is routine. This is a laborious process demanding efforts of time and man-

hours. Here, we have provided our own selection of a potential microbial inoculant

against rice ShB.

9.5.1 Screening of Different PGPR Against ShB Pathogen andSeedling Growth Promotion Under Laboratory Conditions

Seventy PGPR strains that belong to Bacillus, Paenibacillus, Brevibacillus, andArthrobacter were selected from the bacterial culture collection of the Phytopathol-

ogy Laboratory of Auburn University. These PGPR strains were found to be highly

effective in inducing growth-promoting effects in various crops. These strains were

screened against rice ShB pathogen, ShB lesion spread and in promoting rice


seedling growth under laboratory conditions. The mycelial growth inhibition of

R. solani was as high as 83% with B. subtilis MBI 600, compared to the control.

Only four strains completely inhibited the germination of sclerotia. The ShB lesion

spread was determined by highest relative lesion height method (HRLH) and for

effective strain (B. subtilis MBI 600) was found to be only 2.9 as against control

(100). Highest seedling vigor of 13,600 was recorded in comparison to that of control

(4,867) on 10-day-old seedlings. The PGPR strain, B. subtilisMBI 600 was found to

be highly effective in all the screening assays and was selected for further studies.

To further test the efficacy of B. subtilis MBI 600, the strain was produced in a

commercial proprietary liquid formulation by Becker Underwood, Ames, Iowa,

USA. The formulated strain MBI 600 has a proprietary trade name as Integral®. The

product is stored at room temperatures prior to use. The minimum concentration of

Integral in liquid formulation is 2.2 � 1010 cfu ml�1. The details of different

application methods of Integral are shown in Table 9.3.

9.5.2 Efficacy of Integral

To assess the biocontrol suppression of ShB by using antagonistic bacteria and their

combination with fungicide under field conditions for a long time, antagonistic

bacteria and fungicide used to control ShB must be evaluated for durability effect.

Improved plant growth and health by PGPR is either due to direct mechanisms

such as improvement in plant uptake through solubilization of mineral phosphates

Table 9.3 Benefits and use rates of Integral in different crops

Crop Method of

application

Rates of application Target pathogens

Peanut In-furrow 0.1–1.2 fl oz/acre Rhizoctonia, Fusarium,Aspergillus

Cotton, vegetables, soybean

corn

In-furrow 0.1–1.2 fl oz/acre Rhizoctonia, Fusarium

Non-bearing plants (Cherry)

in greenhouses

Soil mix 1.3–13 fl oz/acre Fusarium, Rhizoctonia

Cotton Seed 0.6–2.4 fl oz/100 lb seed Fusarium RhizoctoniaSoybeans Seed 0.13 fl oz/100 lb seed Fusarium RhizoctoniaGreen beans, snap beans,

lima beans, kidney

beans, navy beans,

pinto beans, wax

beans, pole beans,

garden beans, peas,

field beans

Seed 0.6–2.4 fl oz/100 lb seed Fusarium Rhizoctonia

Alfalfa, forage, turf Seed 0.2–12 fl oz/100 lb seed Fusarium RhizoctoniaWheat, barley Seed 0.1–0.6 fl oz/100 lb seed Fusarium RhizoctoniaField corn, sweet corn Seed 0.6–2.4 fl oz/100 lb seed FusariumCanola Seed 1.6–3.8 fl oz/100 lb seed Fusarium Rhizoctonia


and other nutrients (De Freitas et al. 1997; Gaur 1990), nitrogen fixation (Boddey

and Dobereiner 1995), and phytohormone production such as indole 3-acetic acid,

gibberellic acid, cytokinins, and ethylene (Arshad and Frankenberger 1993; Glick

1995). Indirect growth promotion is through biological control of plant pathogens

by producing siderophores (Scher and Baker 1982), antibiotics (Shanahan et al.

1992), hydrogen cyanide (Flaishman et al. 1996), lytic enzymes, and competition

for nutrients and space.

9.5.2.1 In-Vitro Inhibition of ShB Pathogen

The B. subtilisMBI 600 strain of Integral was further characterized for determining

its mode of action against ShB pathogen. The PGPR strain was isolated from the

formulation on TSA and confirmation of its purity was carried out using 16s rDNA

sequence homology and by measuring the 16s rDNA sequence with 1,409 base

pairs of the isolate. The BLAST analysis of the sequencing results confirmed 100%

similarity with B. subtilis. The MBI 600 strain was highly effective against the ShB

pathogen, R. solani and repeatedly shown significant results in inhibiting mycelial

growth (Fig. 9.2) and germination of sclerotia (Fig. 9.3) under in-vitro conditions.

A strong zone of inhibition (3 mm) between mycelial growth of pathogen and

bacterium was observed. Inhibition of sclerotial germination was about 98% at a

concentration of 2.2 � 109 cfu ml�1 whereas at a concentration of

2.2 � 108 cfu ml�1, the inhibition was 37%. Integral was not effective in inhibiting

sclerotial germination at concentrations of 2.2 � 106 and 2.2 � 107 cfu ml�1.

Highest inhibition of sclerotial growth was obtained at a concentration of

2.2 � 109 cfu ml�1 (79%), followed by at 2.2 � 108 cfu ml�1 (72%). Integral

was also effective at lower concentrations with sclerotial growth inhibitions ranging

from 29 to 60% (Table 9.4).

Efficacy of Integral was evaluated in reducing ShB lesions on rice leaves

under in vitro conditions by detached leaf piece assay. Integral concentrations of

Fig. 9.2 Inhibition of mycelial growth of Rhizoctonia solani challenged with Integral


2.2 � 106 through 2.2 � 109 cfu ml�1 were sprayed onto rice leaf pieces (8 cm)

separately. Later the leaves were inoculated with 1-week-old sclerotia of R. solaniat the centre and leaves were incubated in Petri dishes containing moistened filter

papers. ShB lesion length around sclerotium was recorded after 5 days and disease

severity was assessed by highest relative lesion height (HRLH) method. As shown

in Fig. 9.4, Integral at 2.2 � 109 cfu ml�1 significantly reduced ShB lesion spread

on detached rice leaves (4.7) (Table 9.4). At other concentrations, the lesion spread

ranged from 23 to 93 as against untreated control (100).

Integral was tested positive for production of siderophores. However, pro-

duction of IAA, HCN, cellulase, chitinase, and phosphate solubilizing capacity

when tested were negative. Siderophores are low-molecular-weight iron-chelating

agents produced by PGPR that can create iron nutrient competition in soils to

plant pathogens. Since the element iron is present in low quantities in soils,

siderophore production is a strategy by the PGPR to compete with soil-borne

plant pathogens.

Fig. 9.3 Inhibition of sclerotial germination of Rhizoctonia solani by Integral

Table 9.4 Efficacy of Integral on sclerotial germination and sheath blight lesion symptoms of rice

Concentration1 % Inhibition of

sclerotial germination2% Inhibition of sclerotial

growth compared to control3ShB lesion

spread4

2.2 � 106 CFU/ml 0c 28.5d 92.6b

2.2 � 107 CFU/ml 0c 59.5c 71.6c

2.2 � 108 CFU/ml 36.7b 71.8b 22.7d

2.2 � 109 CFU/ml 97.7a 78.8a 4.7e

Control 0c – 99.2a

Means followed by a common letter in the columns are not significantly different at p � 0.051Integral applied at these concentrations to test on sclerotial germination, growth of mycelia, and

suppression of ShB lesions2Sclerotial germination was recorded at 3 days after incubation3Sclerotial growth was recorded at 5 days after incubation and4ShB lesion spread was recorded by Highest Relative Lesion Height method at 7 days after

inoculation


9.5.2.2 Rice Plant Growth Promotion

Seed treatment with Integral was highly effective in promoting rice seedling

development both under laboratory and greenhouse conditions. Under in vitro

conditions, significantly higher root and shoot lengths were observed with Integral

over untreated control. Increase in root and shoot lengths was noticed with an

increase in Integral concentration from 2.2 � 106 cfu ml�1 to 2.2 � 109 cfu ml�1.

As shown in Fig. 9.5, highest root and shoot lengths (47.5 and 39.1 mm, respec-

tively) were recorded at a concentration of 2.2 � 109 cfu ml�1 as against control

(14.3 and 7.6 mm of root and shoot lengths respectively).

Under greenhouse conditions, seed treatment with Integral significantly

improved rice seed germination, seedling emergence, and plant growth. The percent

germination of seeds sown in 15 cm pots filled with field soil was highest at a

concentration of 2.2 � 109 cfu ml�1 (88.9%) as against untreated control (61.1%)

at 7 days after sowing (DAS). Integral application significantly improved root

and shoot lengths at 15 DAS. Highest root length and shoot length were recorded

at a concentration of 2.2 � 109 cfu ml�1 (166 and 335 mm respectively) as against

untreated control (73 and 222 mm respectively) (Fig. 9.6).

9.5.2.3 Chemical Compatibility

Currently, ShB disease management strategy is through use of systemic fungicides

and also with certain non-systemic fungicides (Pal et al. 2005). Pathogen resistance tothese systemic fungicides is of concern, thus demanding integration of PGPR in IDM.

Since, host plant resistance to ShB range only from very susceptible to moderately

susceptible levels in rice (Groth and Bond 2007), use of chemical fungicides has

become a necessary component for an effective ShB management. For effective

functioning of PGPR under the ambit of IDM, their compatibility with commonly

used fungicides and insecticides in rice is also mandatory (Mew et al. 2004).

Fig. 9.4 Suppression of

sheath blight lesions in a

detached leaf assay with

Integral


Combined applications of PGPR with chemical fungicides are an important IDM

package against ShB. Of different PGPR, Pseudomonads and Bacillus spp. werefound to be very effective as a supplement in IDM. Greenhouse and field studies

against rice ShB with different PGPR isolated from farmyard manure, rice seed,

phyllosphere, and rhizosphere proved that three bacteria, P. fluorescens (PF-9),

Bacillus sp. (B-44), and a chitinolytic bacterium (Chb-1) are compatible with

carbendazim at 500 and 1,000 ppm concentrations. Of these, PF-9 was most

effective in reducing ShB severity either alone or in combination with one spray

of 0.1% carbendazim, followed by combination of PF-9 and B-44 (Laha and

Fig. 9.5 Effect of Integral on

rice seed development

Fig. 9.6 Efficacy of Integral

on rice seedling growth


Venkataraman 2001). The B. subtilis (Bs-916) when applied along with jinggang-

mycin was found to colonize the root system effectively. Further, the population

density of BS-916 was maintained in its presence without any further decline (Chen

et al. 2003).

In order to use Integral in ShB management, it has to be compatible with existing

agronomic practices and commonly used chemical fungicides in rice production

systems. In our ongoing research, we have attempted to study our classical product

Integral according to the assays described by Shanmugam and Narayanasamy

(2009) under in vitro conditions. Briefly, in this assay, a loop full of MBI 600

strain onto Nutrient Agar (NA) plates amended with various concentrations

(100–1,000 ppm) of fungicides such as propiconazole, validamycin, benomyl,

carbendazim, tricyclazole, mancozeb, azoxystrobin, and hexaconazole. Plates

were later incubated at room temperature for 48 h and growth of bacterium was

monitored. Further compatibility studies with azoxystrobin and carbendazim were

carried out according to Omar et al. (2006) wherein 100 mL of bacterial inoculum

was added to 250 ml yeast peptone glucose (YPG) liquid medium amended with

fungicides at concentrations at 200 and 400 ppm and incubated on a shaker, growth

of bacterium was enumerated on NA after serial dilution.

Integral exhibited good tolerance to hexaconazole, propiconazole, and valida-

mycin, moderately to tricyclazole and slightly to benomyl and mancozeb at

1,000 ppm. It was highly compatible with carbendazim and azoxystrobin up to

400 ppm whereas complete inhibition was obtained with these fungicides at

800 ppm (Table 9.5). Compatibility to azoxystrobin and carbendazim showed up

to 400 ppm, and good growth of Integral was in carbendazim- and azoxystrobin-

amended medium (Figs. 9.7 and 9.8).

9.5.2.4 Efficacy of PGPR Against ShB Under Greenhouse and Field

Conditions

The time of application of PGPR has significant influence in the management of

ShB disease. Ren et al. (2006) reported that the optimum time of application

Table 9.5 Compatibility of Integral with commonly used fungicides

Fungicides Fungicide concentrations (ppm)a

100 200 400 600 800 1,000

Propiconazole +++ +++ +++ +++ +++ +++

Validamycin +++ +++ +++ +++ +++ +++

Benomyl +++ +++ +++ +++ ++ +

Carbendazim +++ +++ +++ + �� Tricyclazole +++ +++ +++ +++ +++ ++

Mancozeb +++ +++ +++ ++ + +

Azoxystrobin +++ +++ +++ + �� Hexaconazole +++ +++ +++ +++ +++ +++aGrowth of Integral in NA amended with fungicides: +++ ¼ Good; ++ ¼ Moderate; + ¼ Slight;

�� ¼ No growth


of PGPR against ShB under field conditions was during the first day of inoculation

of R. solani. Reduction in ShB severity under field conditions by PGPR is also

dependent on the bacterial concentration. It is interesting to note that the PGPR

when applied as consortia and in conjunction with other fungal antagonists offered

synergistic effect over their individual applications in ShB disease reduction. Talc-

based formulations of two P. fluorescens strains (PF1 and PF7), when applied as

seed, soil, and root dip treatments and foliar sprays, significantly reduced ShB and

leaf-folder incidence under greenhouse and field conditions. The PGPR mixture

proved to be more effective over their individual applications (Radja Commare

et al. 2002). Combined use of P. fluorescens and Trichoderma viride was effective

Fig. 9.7 Compatibility of Integral with Carbendazim. Values are means of five replications.

Means followed by a common letter are not significantly different at p � 0.05

Fig. 9.8 Compatibility of Integral with Azoxystrobin. Values are means of five replications.

Means followed by a common letter are not significantly different at p � 0.05


in rice ShB reduction and enhanced seedling growth (Mathivanan et al. 2006).

Bacillus spp. exhibited synergistic effect when used in conjunction with T. viride(Das et al. 1998) and Gliocladium virens (Sarmah 1999) against ShB. The fermen-

ted product of Bacillus strain Drt-11 when applied in combination with biofungi-

cide, Jinggangmeisu WP (20%) proved to be more effective against ShB over their

individual applications (Chen and Hui 2006).

Our own studies with Integral under greenhouse and field studies effectively

reduced ShB incidence in rice. In a typical greenhouse assay, Integral was evaluated

at concentrations of 2.2 � 106 to 2.2 � 109 cfu ml�1 as seed treatment (ST),

seedling root dip (SD), and foliar sprays (FS). Seed treatment with Integral at

concentrations of 2.2 � 108 and 2.2 � 109 cfu ml�1 significantly improved percent

germination over untreated control. Further, the root and shoot lengths were signifi-

cantly improved (12.2 and 40.7 cm respectively) at 2.2 � 109 cfu ml�1 as against

untreated control (7.9 and 33.8 cm respectively) at 25 DAS. Significant reduction in

ShB severity (9.2 vs. 24.1 in untreated control) (Fig. 9.9), increase in plant height

(73.2 vs. 62.7 cm in untreated control) and number of tillers/plant (11.9 vs. 8.0 in

untreated control) was obtained when Integral was applied at 2.2 � 109 cfu ml�1 as

seed treatment (ST) þ seedling root dip (SD) þ foliar sprays (FS).

Our field studies at Andhra Pradesh Rice Research Institute (APRRI), India

during 2009 indicated significant improvement in root and shoot lengths or rice

seedlings in nursery with Integral seed treatment at 2.2 � 108 and 2.2 �109 cfu ml�1 over untreated control. On a transplanted crop, Integral application as

ST þ SD þ FS at 2.2 � 109 cfu ml�1 significantly reduced sheath blight severity

(19.2 vs. 69.7 in control) and percent diseased tillers (25.1 vs. 99.4 in control)

Fig. 9.9 Suppression of sheath blight severity with Integral under greenhouse conditions


(Fig. 9.10). Plant height (98.1 vs. 78.5 cm in control), tillers/plant (12.8 vs. 10.0

in control), and grain yields were maximum with Integral application at 2.2 �109 cfu ml�1 as ST þ SD þ FS. Integral was also effective in reducing ShB seve-

rity and promoting growth and grain yields at a concentration of 2.2� 108 cfu ml�1.

9.6 Conclusions

As shown by the examples discussed in this chapter, PGPR have good potential in

the management of rice ShB. It is generally believed that the field efficacy of

a particular PGPR strain is dependent on its root colonization capacity. According

to this reasoning, rhizosphere competence of a PGPR strain is a desirable trait for its

effective root colonization and subsequent disease control. Earlier reports indicated

that diversity of PGPR in rice rhizosphere is changing according to soil salinity.

With increase in soil salinity, the population levels of Pseudomonas spp. decreased.In non-saline sites of rice rhizosphere, fluorescent Pseudomonads are the dominant

species whereas in saline sites, these were replaced by salt tolerant species such

as P. alcaligens and P. pseudoalcaligens. Further, organic farming was found to

significantly enhance the diversity of PGPR populations in saline soils (Rangarajan

et al. 2002).

It should be noted that although rhizosphere competence is considered important

for effective PGPR biocontrol agents, there could be exceptions. For example, if a

particular PGPR-based product is applied as a foliar spray, rhizosphere colonization

would not be strictly required. For example, with the product Intergral, which was

highly effective in biocontrol of ShB in field trials in India, one application method

was a foliar spray. To date, there are no published studies examining possible

rhizosphere competence or root colonization on rice by the PGPR strain contained

in Integral (B. subtilis strain MBI600).

Fig. 9.10 Sheath blight severity under field conditions


Several biotic and abiotic factors also have significant impact on the field

consistency of a formulated PGPR strain in rice ShB management. Since gram-

positive bacilli produce endospores that can withstand desiccation and have a long

shelf life, they are considered to be ideal candidates for commercial use against

ShB. Fungicidal compatibility of selected PGPR strain is another important factor

that determines the efficacy of PGPR under IDM. Consistent efforts are therefore

needed to select PGPR strain with all the desirable traits that contribute to effective

rice ShB management.

Although several advantages have been reported with the use of microbial

inoculants in rice, variability in effectiveness of field performance remains a con-

straint. To overcome this, comprehensive basic research is essential in the areas

of selection of microbial agents that focus on identifying strains that occupy the

same ecological niche with that of pathogens such as roots, the phylloplane, and

vascular systems. Application of novel techniques such as PCR, RFLP, and RAPD

for rapid identification of bacterial strains with desirable traits like biocontrol and

growth-promoting mechanisms are therefore necessary. Integrating these basic

research concepts with studies on greenhouse and field studies are therefore essen-

tial before devising IDM approaches for ShB management in rice. The PGPR that

are identified in these respects should be maintained as important genetic resource,

which will be useful for future studies that form an alternate to the presently

available chemical control of ShB.

Acknowledgments The primary author is thankful to the authorities of Acharya N G Ranga

Agricultural University, India for granting sabbatical to pursue his Ph.D. program at Auburn

University, USA. The authors are thankful to the Associate Director of Research, A. P. Rice

Research Institute, India for extending help in carrying out greenhouse and field studies. The

support of Rice Research Station, LSU AgCenter, USA in providing the seed material and

pathogen for our investigations, and of Becker Underwood, USA in providing the Integral

formulation is highly appreciated.

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